JP6383359B2 - CRZ1 mutant fungal cells - Google Patents

Description

This application claims the benefit of US Provisional Patent Application No. 61 / 716,670, filed Oct. 22, 2012; the provisional application is incorporated herein by reference in its entirety. Is done.

  The present invention, in conjunction with its method of use, relates to CRZ1 mutant alleles and fungal host cells such as Pichia pastoris containing the alleles.

GlycoFi has engineered Pichia to produce recombinant glycoproteins with human-like glycosylation. However, extensive genetic recombination has also caused fundamental changes in cell wall structure, making it easier to lyse these sugar engineered strains during fermentation and to reduce cell robustness (robustness). These undesirable traits resulted in a substantial decrease in cell viability and a significant increase in intracellular protease leakage into the fermentation broth, resulting in a decrease in both the yield and quality of the recombinant product. Isolated fungal host cells, such as Candida albicans; Hansenula polymorpha; Schizosaccharomyces pombe; Saccharomyces cerevisia; It is known that it lacks functional OCH1, a polypeptide in the fungal glycosylation pathway, and is temperature sensitive. For example, the Candida albicans och1 disruption strain is temperature sensitive at 42 ° C. (Bates et al., Outer Chain N-Glycans Are required for Cell Wall Integrity and Virulence chain). Candida albicans is required for cell wall integrity and virulence), The Journal of Biological Chemistry 281: 90-98 (2006)); Hansenula polymorpha oc1 disruption strain at 45 ° C. Yes (Kim et al., Functional Charac terization of the Hansenula polymorpha HOC1, OCH1, and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family involved in Protein Glycosylaton (as a member of the yeast OCH1 mannosyltransferase family involved in the glycosylation of the protein, Hansenula polymorpha HOC1, OCH1 and OCR1 Functional characterization of genes), The Journal of Biological Chemistry, 281: 6261-6272 (2006)); Schizosaccharomyces pombe och1 disrupted strain is temperature sensitive at ^{37 ℃ (Yoko-o et al} ., Schizosaccharomyces pombe och1 + encodes alpha-1,6-Mmannosyltransferase that is involed in outer chain elongation of N-linked oligosaccharides ( Schizosaccharomyces-cylinder och1 ⁺ Strain encodes alpha-1,6-mannosyltransferase involved in the outer chain extension of N-linked oligosaccharides), FEBS Letters 489: 75-80 (2001)); Saccharomyces cerevisiae) och1 disruption The strain is temperature sensitive at 37 ° C (N kayama et al. , OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides (OCH1 is a novel membrane bound mannosyltransferase O 11 (7): 2511-9 (1992)); and the Pichia pastoris och1 disruption strain is temperature sensitive at 37 ° C. (Choi et al., Use of combinatorial ligands to humanisin the yeast Pichia pastoris (use of a combinatorial gene library for humanizing N-linked glycosylation in the yeast Pichia pastoris), Proc Natl Acad Sci USA. 100 (9): 5022-7 (2003)). In cell culture, och1 ^- fungal host cells (e.g., Pichia (Pichia) cells) further genetic manipulation to more robustness to would be worth.

  An unexpected candidate for genetic manipulation to enhance the fermentation robustness of Pichia is CRZ1, a zinc finger transcription factor. CRZ1 has several S.P. It is known to regulate S. cerevisiae plasma membrane and cell wall regulatory genes (Cyert, Biochemical and Biophysical Research Communications 311: 1143-1150 (2003)). The disruption of plasma membrane and cell wall synthesis due to CRZ1 mutation would have been expected to make Pichia cells weaker and more robust. S. Published characterization of S. cerevisiae CRZ1 will be less viable and robust when Pichia cells lacking functional CRZ1 are placed under high temperature stress Would have led those skilled in the art to anticipate. (Matheos et al., Genes & Development 11: 3445-3458 (1997); Stathopoulos et al., Genes & Development 11: 3432-3444 (1997)).

  The present invention provides an isolated fungal host cell that lacks a functional CRZ1 polypeptide (eg, Pichia, such as Pichia pastoris), for example, wherein the cell comprises a functional CRZ1 Show enhanced fermentation robustness and increased production of heterologous polypeptides such as immunoglobulins, for example, where endogenous CRZ1 is mutated, disrupted or partially or completely deleted Optionally comprising a heterologous polynucleotide encoding a heterologous polypeptide such as an immunoglobulin (eg, operably linked to a promoter such as a methanol-inducible promoter). In one embodiment of the present invention, (i) endogenous CRZ1 is L33 → STOP; Q214 → STOP; L294 → STOP; S298 → STOP; E403 → G; F406 → S; F406 → L; C411 → F; and K469 → N, destruction of endogenous CRZ1, complete deletion of endogenous CRZ1, partial deletion of endogenous CRZ1 (eg, 33aa-terminal, 214aa-terminal, 294-terminal, 298-terminal of CRZ1 polypeptide deleted Encodes a polypeptide comprising one or more mutations selected from the group consisting of; or (ii) endogenous CRZ1 is a1407c; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t; including one or more mutations selected from the group consisting of t98a; Others (iii) Endogenous CRZ1 does not encode a functional C-terminal zinc finger domain. In one embodiment of the invention, the isolated fungal host cell also has one or more of the following characteristics (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, (I) where one or more endogenous beta-mannosyltransferase genes are mutated, disrupted, truncated, or partially or wholly deleted; (ii) alpha A polynucleotide encoding -1,2 mannosidase, alpha-1,3 mannosidase or alpha-1,6 mannosidase; (iii) wherein one or more endogenous phosphomannosyltransferases are mutated, disrupted, truncated Or (iv) a single subunit oligosaccharyltransferase ( For example, Leishmania sp. STT3D); (v) where endogenous dolichol phosphate mannose-dependent alpha-1,3-mannosyltransferase (eg, ALG3) is mutated, disrupted, Truncated or partially or wholly deleted; (vi) comprises a polynucleotide encoding endomannosidase; (vii) a bifunctional UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase; Comprising one or more polynucleotides encoding N-acetylneuraminic acid-9-phosphate synthase or CMP-sialic acid synthase; (viii) wherein the endogenous ATT1 gene is mutated, disrupted, truncated or Partially or completely deleted (Ix) where alpha-1,6-mannosyltransferase (eg, OCH1) is mutated, disrupted, truncated, or partially or completely deleted; (x) galactosyltransferase Including, for example, alpha 1,3-galactosyltransferase or beta 1,4-galactosyltransferase; (xi) including a sugar nucleotide transporter, such as the UDP-galactostotransporter (DmUGT); (xii) a sialyltransferase, such as Alpha-2,6-sialyltransferase (MmST6-33); (xiii) acetylglucosaminyltransferase, eg, GNT1 or GNT2 or GNT4; and / or (xiv) where Other more endogenous protease genes (e.g., PEP4 and PRB1) are mutated, it is deleted destroying or partially or completely deleted. The present invention also includes introducing into a cell a heterologous polynucleotide that recombines homologously with endogenous CRZ1 and that partially or completely deletes or destroys endogenous CRZ1. A method for producing an isolated fungal host cell is provided along with a fungal host cell produced by the method.

  The present invention also includes, for example, the nucleotide sequence of SEQ ID NO: 2 comprising a mutation selected from the group consisting of a1407c,; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t and t98a, L33 → STOP; → STOP; L294 → STOP; S298 → STOP; E403 → G; F403 → S; F406 → L; including the amino acid sequence shown in SEQ ID NO: 3 comprising a mutation selected from the group consisting of C411 → F and K469 → N Also provided is an isolated polynucleotide encoding the polypeptide. An isolated vector containing the polynucleotide also forms part of the present invention. Isolated polypeptides encoded by the nucleotides also form part of the present invention.

The present invention also encodes a polypeptide from the group consisting of L33 → STOP; Q214 → STOP; L294 → STOP; S298 → STOP; E403 → G; F406 → S; F406 → L; C411 → F and K469 → N An isolated crz1 ^mutant fungal host cell (eg, Pichia) with improved viability at elevated temperatures (eg, 32 °) comprising introducing a selected mutation into an endogenous CRZ1 gene in the fungal cell A method for creating Pichia, such as Pichia pastoris, is also provided.

The present invention also provides (i) a polynucleotide encoding the heterologous polypeptide wherein the isolated crz1 ^mutant fungal host cell (eg, Pichia pastoris such as Pichia pastoris) (eg, the present And (ii) culturing the host cell under conditions favorable for expression of the heterologous polypeptide in the cell (eg, at 24 ° C.); and (iii) Also provided is a method of producing one or more heterologous polypeptides (eg, immunoglobulin polypeptides) comprising isolating the heterologous polypeptide from the host cell. In one embodiment of the invention, the heterologous polynucleotide encoding the heterologous polypeptide is operably linked to a methanol-inducible promoter, and the isolated fungal host cell is in the presence of methanol. Culturing is performed under conditions favorable for expression of the heterologous polypeptide.

A thermostable mutant exhibiting improved fermentation robustness and Fc titer. A series of mutagenized Pichia pastoris strains. A series of mutagenized Pichia pastoris strains. A series of mutagenized Pichia pastoris strains. N-glycan profile of thermostable mutants. G0 = N-glycan terminated with GlcNac; G1 = N-glycan terminated with single galactose; G2 = N-glycan terminated with double galactose; A1 = N-glycan terminated with single sialic acid; A1H = A1 hybrid, N-glycan terminated with a single sialic acid having a hybrid structure; A2 = N-glycan terminated with double sialic acid; M5 = Man ₅ ; M6 + = Man ₆₊ . Large-scale sequencing of thermostable mutants with multiple mutations in Pp02g02g02120 (CRZ1) and two mutations in Pp01g00680 (ATT1) corresponding to the enclosed “+” sign. The mutein Pp02g02120 was homologous to the Saccharomyces cerevisiae CRZ1 zinc finger transcription factor involved in stress response. Various isolated mutant PpCRZ1 alleles are shown. “ ^* ” Indicates the position of the displayed mutation. Plasmid pGLY12829. Plasmid pGLY12832. DNA construct constructed for Pichia pastoris CRZ1 deletion and truncation. Pichia pastoris CRZ1 deletion and truncation mutants showed improved fermentation robustness at 32 ° C. Pichia pastoris CRZ1 deletion improves fermentation robustness in att1 mutants at 34 ° C.

Detailed Description of the Invention

  Contrary to expectation, Pichia pastoris cells lacking functional CRZ1 showed improved thermostability and enhanced robustness.

  Random mutagenesis was performed to broadly improve the characteristics of the strain and several Pichia pastoris strains, heat-resistant mutants with significantly improved fermentation robustness, were identified. Non-mutagenized glycoengineered parent strains show a temperature-sensitive phenotype (Choi et al. 2003) and survive 40 to 60 hours after induction at 32 ° C, whereas all mutant strains at 32 ° C It lasted 90 to 110 hours after induction. This extended induction period significantly improved the yield and properties of recombinant proteins expressed in these thermostable strains. In order to identify the mutation responsible for this increased thermotolerance and fermentation robustness, genome sequencing was performed on nine independently isolated mutants and a separate open for each mutant. Non-synonymous mutations within the reading frame (ORF) were confirmed. Surprisingly, all nine mutants contained a distinct mutation within the coding region of one gene, Pichia pastoris CRZ1. More importantly, the Pichia pastoris CRZ1 mutation was the only non-synonymous single base diversity detected in the three mutants YGLY29010, YGL29031 and YGL29042. Taken together, these genomic sequencing results indicate that mutations in the Pichia pastoris CRZ1 gene are responsible for the thermostable and fermentation robust phenotype. In addition, non-mutagenized glycoengineered strains in which endogenous CRZ1 was mutated and thus lacking a functional CRZ1 protein also showed viability of 90-110 hours after induction at 32 ° C.

“CRZ1 ^wt ” fungal host cells contain wild-type CRZ1.

  “PpCRZ1” is Pichia pastoris CRZ1.

  “ScCRZ1” is Saccharomyces cerevisiae CRZ1.

  The elevated temperature for growth of isolated fungal cells such as Pichia, for example Pichia pastoris, is 28 ° C., 29 ° C. or 30 ° C. or higher, for example 32 ° C.

  A heterologous polynucleotide is a polynucleotide that is introduced into a fungal host cell and encodes a heterologous polypeptide. For example, the heterologous polynucleotide can be an immunoglobulin heavy chain or an immunoglobulin light chain, eg, a light chain or heavy chain variable domain, and optionally, eg, a fully human antibody, eg, derived from an antibody or antigen-binding fragment thereof, Antibody constants derived from antigen-binding fragments of antibodies such as humanized antibodies, chimeric antibodies, bispecific antibodies, Fab antibody fragments, F (ab) 2 antibody fragments, Fv antibody fragments, single chain Fv antibody fragments or dsFv antibody fragments Can code a domain. None of these antibodies are insulin-like growth factor receptor 1, VEGF, interleukin-6 (IL6), IL6 receptor, respiratory rash virus (RSV), CD20, tumor necrosis factor alpha, NF kappa B activation receptor It can specifically bind to any epitope such as a ligand (RANKL) or the RANKL receptor RANK, IgE, Her2, Her3 or epidermal growth factor receptor.

  An “endogenous” gene is a chromosomal copy of the gene.

A glossary of gene names that may be mentioned herein is as follows:
ScSUC2 S. S. cerevisiae invertase OCH1 alpha-1,6-mannosyltransferase K1MNN2-2-2 Lactis (K. lactis) UDP-GlcNAc transporter Sugar nucleotide transporter BMT1 beta-mannose-transfer (beta-mannose removal) beta-mannosyltransferase BMT2 beta-mannose-transfer (beta-mannose removal)
BMT3 beta-mannose transfer (beta-mannose removal)
BMT4 beta-mannose transfer (beta-mannose removal)
MNN4L1 MNN4 like 1 (charge removal)
MmSLC35A3 UDP-GlcNAc transporter mouse homolog PNO1 Phosphomannosylation of N-linked oligosaccharides (charge removal)
MNN4 mannosyltransferase (charge removal)
ScGAL10 UDP-glucose 4-epimerase XB33 Truncated HsGalT1 fused to ScKRE2 leader
Truncated DmMNSII fused to DmUGT UDP-galactose transporter KD53 ScMNN2 leader
Truncated RnGNTII fused to TC54 ScMNN2 leader
Truncated HsGNTI fused to NA10 PpSEC12 leader
Truncated MmM NS1A fused to FB8 ScSEC12 leader
MmCST Mouse CMP-sialic acid transporter HsGNE Human UDP-GlcNAc2-epimerase / N-acetylmannosamine kinase HsCSS Human CMP-sialic acid synthase HsSPS Human N-acetylneuraminic acid-9-phosphate synthase MmST6-33 ScKRE2 leader Fused truncated mouse alpha-2,6-sialyltransferase TrMDS1 secreted T. pylori T. reesei MNS1
STE13 Golgi dipeptidylaminopeptidase DAP2 Vacuolar dipeptidylaminopeptidase ALG3 Dolichol phosphate mannose-dependent alpha (1-3) mannosyltransferase STT3D Oligosaccharyltransferase CiMNS1 Coccidioides-imitis mannosidase I
Molecular Biology In the present invention, conventional molecular biology, microbiology and recombinant DNA techniques can be used within the technical scope of the field. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In general, the terminology and techniques used herein in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization are well known in the art. And are commonly used. The methods and techniques of the present invention are described according to conventional methods well known in the art and as described in various general and more specific references, cited and discussed throughout this specification, unless otherwise indicated. Generally implemented. For example, James M.M. Cregg (Editor), Pichia Protocols (Methods in Molecular Biology) (Pichia's protocol (method of molecular biology)), Humana Press (2010), Sambrook et al. Molecular Cloning: A Laboratory Manual (Molecular Cloning: Experimental Manual), 2nd ed (2nd edition). , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. (1989); Ausubel et al, Current Protocols in Molecular Biology (Current Protocol of Molecular Biology), Greene Publishing Associates (1992, and Supplements to 2002, AL); Antibody: Experimental Manual), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. (1990); Taylor and Drickkamer, Introduction to Glycobiology (Introduction to Glycobiology), Oxford Univ. Press (2003); Worthington Enzyme Manual (Worthington Enzyme Manual), Worthington Biochemical Corp. , Freehold, N .; J. et al. ; Handbook of Biochemistry: Section A Proteins, Vol 1 (Biochemistry Handbook: Section A Protein, Volume 1), CRC Press (1976); Animal Cell Culture (Animal Cell Culture) (RI Freshney, ed. (Ed.) (1986)); Immobilized Cells And Enzymes (immobilized cells and immobilized enzymes) (IRL Press, (1986)); See Perbal, A Practical Guide To Molecular Cloning (1984).

  "Polynucleotide" or "nucleic acid" includes single and double stranded forms or other forms of DNA and RNA.

A “polynucleotide sequence” or “nucleotide sequence” is a series of nucleotide bases (also referred to as “nucleotides”) in a nucleic acid, such as DNA or RNA, and means a series of two or more nucleotides. Any polynucleotide comprising the nucleotide sequence set forth herein (eg, crz1 ^mutant ) forms part of the present invention.

  A “coding sequence” such as RNA or polypeptide or a sequence that “encodes” an expression product is a nucleotide that, when expressed, results in the production of the product (eg, a heterologous polypeptide such as an immunoglobulin heavy chain and / or light chain). A sequence (eg, heterologous nucleotide).

As used herein, the term “oligonucleotide” refers to a nucleic acid, generally only about 100 nucleotides (eg, 30, 40, 50, 60, 70, 80 or 90) that can hybridize to a polynucleotide molecule. Represents. Oligonucleotides can be labeled, for example, by incorporation of nucleotides covalently linked to a label such as ³² P-nucleotide, ³ H-nucleotide, ¹⁴ C-nucleotide, ³⁵ S-nucleotide or biotin.

A “protein”, “peptide” or “polypeptide” (eg, a heterologous polypeptide such as an immunoglobulin heavy chain and / or light chain) comprises a linked sequence of two or more amino acids. Any polypeptide comprising an amino acid sequence set forth herein (eg, a Crz1 ^mutant polypeptide) forms part of the present invention.

  A “protein sequence”, “peptide sequence” or “polypeptide sequence” or “amino acid sequence” represents a series of two or more amino acids in a protein, peptide or polypeptide.

The term “isolated polynucleotide” or “isolated polypeptide” is partially or completely separated from other components or any other contaminants normally found in cells or in recombinant DNA expression systems. Each polynucleotide or polypeptide is included. These components include, but are not limited to, cell membranes, cell walls, ribosomes, polymerases, serum components and exogenous genomic sequences. The scope of the present invention includes an isolated polynucleotide (eg, crz1 ^mutant ) set forth herein and an isolated polypeptide encoded by the polynucleotide.

  An isolated polynucleotide or polypeptide is preferably a substantially homogeneous composition of molecules, but may contain some heterogeneity.

  “Amplification” of DNA, when used, includes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR, see Saiki, et al. , Science (1988) 239: 487.

In general, a “promoter” or “promoter sequence” is a transcription of a coding sequence that binds RNA polymerase in a cell (eg, directly or by other promoter-binding protein or substance) and to which it is operably linked. Is a DNA regulatory region capable of initiating A crz1 ^mutant polynucleotide operably linked to a promoter forms part of the present invention. An isolated crz1 ^mutant fungal host cell comprising a heterologous polynucleotide operably linked to a promoter (eg, encoding an immunoglobulin polypeptide) also forms part of the present invention.

  Coding sequences (eg, heterologous polynucleotides, eg, reporter genes or immunoglobulin heavy and / or light chain coding sequences) “operably linked” to transcriptional and translational regulatory sequences (eg, promoters of the invention). , "Under the control of", "functionally related to" or "operably related to" when the sequence is mediated by RNA polymerase mediated RNA of the coding sequence, preferably mRNA Directing transcription, then the RNA can be RNA spliced (if it contains an intro) and optionally translated into the protein encoded by the coding sequence.

The present invention includes a vector or cassette comprising a crz1 ^mutant polynucleotide. Vectors containing heterologous polynucleotides encoding heterologous polypeptides can also be used in various crz1 ^mutant fungal host cells for the production of heterologous polypeptides (eg, immunoglobulins). The term “vector” includes a DNA or RNA sequence introduced into the host cell to transform the host and, optionally, to promote expression and / or replication of the introduced sequence. Vehicles (eg, plasmids) that can be made are included. Vectors suitable for use herein include plasmids, integratable DNA fragments and other vehicles that can facilitate the introduction of nucleic acids into the genome of a host cell (eg, Pichia pastoris). included. Although plasmids are the most commonly used form of vector, all other forms of vectors that provide similar functions and are known or become known in the art are described herein. Suitable for use. For example, Pouwels, et al. , Cloning Vectors: A Laboratory Manual (Cloning Vector: Experimental Manual), 1985 and Supplements (Addendum), Elsevier, N .; Y. , And Rodriguez et al. (Eds (ed)), Vectors: A Survey of Molecular Cloning Vectors and Ther Uses (Vector: Overview of Molecular Cloning Vectors and Their Use), 1988, Buttersworth, Boston, MA. The vector optionally includes a secretion signal (eg, alpha mating factor (α-MF) pre-pro leader sequence) operably linked to the heterologous polynucleotide. An isolated crz1 ^mutant fungal host cell comprising a vector containing, for example, a heterologous polynucleotide operably linked to a promoter (eg, a polynucleotide encoding an immunoglobulin polypeptide) is also part of this invention. Make it.

  A polynucleotide operably linked to a promoter (eg, a heterologous polynucleotide, eg, a heterologous polynucleotide encoding an immunoglobulin heavy chain and / or light chain) can be expressed in an expression system. The term “expression system” means host cells and compatible vectors capable of expressing a protein or nucleic acid carried by the vector and introduced into the host cell under suitable conditions. Common expression systems include fungal host cells (eg, Pichia pastoris) and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors.

The term “methanol induction” increases expression of a polynucleotide (eg, a heterologous polynucleotide) operably linked to a methanol-inducible promoter in a host cell of the invention by exposing the host cell to methanol. Represents that. A crz1 ^mutant containing a polynucleotide operably linked to a methanol-inducible promoter forms part of the present invention. A method for inducing expression of a heterologous polynucleotide fused to a methanol-inducible promoter by exposing crz1 ^mutant fungal cells comprising the promoter construct to methanol, and under conditions favorable for expression of the encoded heterologous polypeptide The method for culturing the cells forms part of the present invention.

  The following references regarding the BLAST algorithm are incorporated herein by reference: BLAST ALGORITHMS (BLAST algorithm): Altschul, S .; F. , Et al. , J .; Mol. Biol. (1990) 215: 403-410; Gish, W .; , Et al. , Nature Genet. (1993) 3: 266-272; Madden, T .; L. , Et al. , Meth. Enzymol. (1996) 266: 131-141; Altschul, S .; F. , Et al. , Nucleic Acids Res. (1997) 25: 3389-3402; Zhang, J. et al. , Et al. Genome Res. (1997) 7: 649-656; Woughton, J. et al. C. , Et al. , Comput. Chem. (1993) 17: 149-163; Hancock, J. et al. M.M. , Et al. , Comput. Appl. Biosci. (1994) 10: 67-70; ALIGNMENT SCORING SYSTEMS (alignment scoring system): Dayhoff, M .; O. , Et al. , “A model of evolutionary change in proteins.” In Atlas of Protein Sequence and Structure (“Models of Evolutionary Changes in Proteins in the Sequence of Protein Sequences and Structures”), (1978) vol. 5, suppl. 3 (Appendix 3). M.M. O. Dayhoff (ed), pp. 345-352, Natl. Biomed. Res. Found. , Washington, DC; Schwartz, R .; M.M. , Et al. , “Matrix for detecting distant relations.” In Atlas of Protein Sequence and Structure (“Matrix for Distal Relationship Detection” in protein sequence and structure charts), (1978) vol. 5, suppl. 3 (Appendix 3). “M.O. Dayhoff (ed), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. Biol. (1991) 219. States, DJ, et al., Methods (1991) 3: 66-70; Henikoff, S., et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10915. Altschul, SF, et al., J. Mol.Evol. (1993) 36: 290-300; .Sci.USA ( 1990) 87: 2264-2268; Karlin, S., et al., Proc. Natl. Acad. Sci. USA (1993) 90: 5873-5877 ;, Dembo, A., et al., Ann. Prob. (1994). ) 22: 2022-2039; and Altschul, SF “Evaluating the statistical signature of multiple distributive alignments. “In Theoretical and Computational Methods in Genome Research (“ Evaluation of statistical significance of multiple distinct local alignments ”in theoretical and computational methods in genomic studies) (S. Suhai, ed (ed.).) (1997). ) Pp. 1-14, Plenum, New York.

CRZ1
In addition to isolated fungal host cells comprising endogenous CRZ1 mutated in the method (eg, with mutations, partial deletions or total deletions or gene disruptions), the present invention provides mutations (crz1 ^mutant polynucleotides). ) Isolated CRZ1 polynucleotide and a polypeptide encoded by the polynucleotide (crz1 ^mutant polypeptide). Specific examples of such mutations in the Pichia pastoris CRZ1 polynucleotide are sequences having one or more of the following mutations: a1407c; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t; A polynucleotide comprising the nucleotide sequence of number 2. These CRZ1 polynucleotides have the following mutations: L33 → STOP (stop codon); Q214 → STOP; L294 → STOP; S298 → STOP; E403 → G; F406 → S; F406 → L; C411 → F; → encodes a CRZ1 polypeptide having the amino acid sequence of SEQ ID NO: 3 having one or more of N. The mutant polynucleotide can be introduced into the endogenous CRZ1 chromosomal locus to replace the wild-type endogenous CRZ1 with the mutant CRZ1.

  The invention encompasses any CRZ1 polynucleotide comprising a mutation encoding a CRZ1 polypeptide that lacks a functional C-terminal zinc finger domain (eg, a nonsense mutation or a deletion that truncates the zinc finger domain). Such polypeptides are also within the scope of the present invention.

  The zinc finger domain of the Pichia pastoris CRZ1 polypeptide has an amino acid sequence: SIYACSLCSKRFTRPYNLK SHLRTHADERPFQCSICGKAFARSHDRKRHEDLHSGERKYCCKGVLSDGVTTWGCEKRFARTDALGRHFKTECGKLC (amino acids 376-471 of SEQ ID NO: 3).

本発明は、ピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）の突然変異株およびサッカロミセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｄｅｓ ｃｅｒｅｖｉｓｉａｅ）ＣＲＺ１ポリペプチドおよび当該ポリペプチドをコードするポリヌクレオチドを含む。ピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）およびサッカロミセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＣＲＺ１ポリペプチドの具体例は、上に示すＢＬＡＳＴＰ比較において^★または＾で特記した位置に、配列番号１に示すアミノ酸配列に対する一または複数の変化を含む。 The BLASTP comparison between the Saccharomyces cerevisiae CRZ1 polypeptide (SEQ ID NO: 1) and the Pichia pastoris CRZ1 polypeptide (SEQ ID NO: 3), confirmed in the random mutation screen, is as follows: is there:

The invention includes mutants of Pichia pastoris and Saccharomyces cerevisiae CRZ1 polypeptides and polynucleotides encoding the polypeptides. Specific examples of Pichia pastoris and Saccharomyces cerevisiae CRZ1 polypeptides can be found in one or more of the amino acid sequences shown in SEQ ID NO: 1 at the positions indicated by ^★ or ^ in the BLASTP comparison shown above. Includes changes.

  The homology of CRZ1 is known in the art. Specific examples of CRZ1 are shown below. In one embodiment of the invention, the Saccharomyces cerevisiae CRZ1 polypeptide or the Pichia pastoris CRZ1 polypeptide is at least about 90% (eg, 90%) relative to SEQ ID NO: 1 or 3, respectively. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence similarity or sequence homology. In one embodiment of the invention, the Pichia pastoris CRZ1 polynucleotide is at least about 90% (eg, 90%, 91%, 92%, 93%, 94%, 95%) relative to SEQ ID NO: 2. 96%, 97%, 98% or 99%) sequence homology.

（配列番号１）
ピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）ＣＲＺ１野生型オープンリーディングフレーム

（配列番号２）
ピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）ＣＲＺ１野生型ポリペプチド

（配列番号３）
宿主細胞
本発明は、その遺伝的背景にさらなる突然変異を含有し得る単離されたｃｒｚ１^{ｍｕｔａｎｔ}真菌宿主細胞を含む。「ｃｒｚ１^{ｍｕｔａｎｔ}」真菌宿主細胞において（半数体にせよ二倍体にせよ）、内在性の染色体ＣＲＺ１遺伝子が、突然変異、破壊または部分的にまたは完全に欠失されているか、またはＣＲＺ１タンパク質の発現が、何らの方法で（例えば、アンチセンスＲＮＡ、低分子干渉ＲＮＡ（ＳｉＲＮＡ）等の干渉ＲＮＡによって）低減されているか、またはＣＲＺ１ポリペプチドの活性が、化学的に（例えば、小分子阻害剤によって）不活性化されていて、従って該細胞は、ＣＲＺ１が突然変異または干渉等されなかった単離真菌宿主細胞と比較して、どんな程度にでも機能的ＣＲＺ１ポリペプチド濃度および／またはＣＲＺ１活性を部分的にまたは完全に欠く。本発明の一実施形態において、該ｃｒｚ１^{ｍｕｔａｎｔ}真菌宿主細胞は、高温または２４℃において、完全なレベルのＣＲＺ１を含む真菌宿主細胞よりも（例えば、ファーメンターまたはバイオリアクターにおいて）生存可能であり，例えば３２℃において、例えば３２℃での誘導の約９０〜１１０時間までのあいだ生存可能である。本発明の一実施形態において、例えば、異種Ｆｃポリペプチドをコードする異種ポリヌクレオチドを含む単離されたｃｒｚ１^{ｍｕｔａｎｔ}真菌宿主細胞は（例えば、ファーメンターまたはバイオリアクターにおいて）、Ｆｃポリペプチドをコードする当該異種ヌクレオチドを含むＣＲＺ１野生型真菌宿主細胞よりも、顕著に多くの異種ポリペプチド（例えば、４倍または５倍以上）、例えば、Ｆｃポリペプチドを発現する（当該ｃｒｚ１^{ｍｕｔａｎｔ}真菌宿主細胞は本発明の範囲内である）。本発明の一実施形態において、例えば、異種Ｆｃポリペプチドをコードする異種ポリヌクレオチドを含む単離されたｃｒｚ１^{ｍｕｔａｎｔ}真菌宿主細胞は（例えば、ファーメンターまたはバイオリアクターにおいて）、ＣＲＺ１^ｗｔ真菌宿主細胞のＮ‐グリカンプロファイルと実質的に同一のＮ‐グリカンプロファイルを有する、例えば、効率的にそのＮ‐グリカン、例えば、高レベルの末端シアル酸を有し、および／または７７から８４％の範囲のＡ２レベル、および／または４から７％のＡ１レベルを有する、ＦｃＮ‐グリカンを効率的に修飾することのできる異種ポリペプチド、例えば、Ｆｃポリペプチドを発現する（当該ｃｒｚ１^{ｍｕｔａｎｔ}真菌宿主細胞は、本発明の範囲内である）。 Saccharomyces cerevisiae CRZ1 wild type polypeptide

(SEQ ID NO: 1)
Pichia pastoris CRZ1 wild type open reading frame

(SEQ ID NO: 2)
Pichia pastoris CRZ1 wild type polypeptide

(SEQ ID NO: 3)
Host cells The present invention includes isolated crz1 ^mutant fungal host cells that may contain additional mutations in their genetic background. In a “crz1 ^mutant ” fungal host cell (whether haploid or diploid), the endogenous chromosomal CRZ1 gene is mutated, disrupted or partially or completely deleted, or the expression of CRZ1 protein Is reduced in any way (eg, by interfering RNA, such as antisense RNA, small interfering RNA (SiRNA)), or the activity of the CRZ1 polypeptide is chemically (eg, by a small molecule inhibitor) ) Inactivated, so that the cell partially divides functional CRZ1 polypeptide concentration and / or CRZ1 activity to any extent compared to an isolated fungal host cell in which CRZ1 was not mutated or interfered with, etc. Or completely missing. In one embodiment of the present invention, the crz1 ^mutant fungal host cell is more viable (eg, in a fermenter or bioreactor) than a fungal host cell containing complete levels of CRZ1 at elevated temperatures or 24 ° C., for example At 32 ° C., for example, it is viable for up to about 90-110 hours of induction at 32 ° C. In one embodiment of the invention, for example, an isolated crz1 ^mutant fungal host cell comprising a heterologous polynucleotide encoding a heterologous Fc polypeptide (eg, in a fermenter or bioreactor) Expresses significantly more heterologous polypeptides (eg, 4 times or more than 5 times), eg, Fc polypeptides, than the CRZ1 wild type fungal host cells containing the heterologous nucleotides (the crz1 ^mutant fungal host cells of the invention Is within range). In one embodiment of the invention, for example, an isolated crz1 ^mutant fungal host cell comprising a heterologous polynucleotide encoding a heterologous Fc polypeptide (eg, in a fermenter or bioreactor) is the N of CRZ1 ^wt fungal host cell. An N-glycan profile that is substantially identical to the glycan profile, eg, the N-glycan, eg, having a high level of terminal sialic acid, and / or an A2 level in the range of 77 to 84% And / or expresses a heterologous polypeptide capable of efficiently modifying FcN-glycans having an A1 level of 4 to 7%, such as an Fc polypeptide (the crz1 ^mutant fungal host cell of the invention Is within range).

In one embodiment of the invention, the isolated crz1 ^mutant fungal host cell of the invention comprises an endogenous mutant CRZ1 polypeptide, eg, it contains the following mutations: L33 → STOP; Q214 → STOP; L294 → STOP S403 → STOP; E403 → G; F406 → S; F406 → L; C411 → F; and K469 → N, comprising the amino acid sequence of SEQ ID NO: 3; for example, in one embodiment of the invention: The mutated endogenous CRZ1 polynucleotide of the isolated crz1 mutant fungal host cell comprises nucleotides of SEQ ID NO: 2 having one or more of the following mutations: a1407c; g1232t; t1216c; t1217c; a1208g; c893a; Contains an array. In one embodiment of the invention, in an isolated crz1 ^mutant fungal host cell of the invention, endogenous CRZ1 is mutated so that it lacks a functional C-terminal zinc finger domain by mutation or truncation. In one embodiment of the present invention, the endogenous CRZ1 of the fungal host cell is a mutant CRZ1, such as a nonsense mutant, deletion mutant, total deletion mutant or nonsense mutation shown herein. It was replaced with any of CRZ1, such as mutants including mutants containing mutations.

The endogenous CRZ1 gene in an isolated crz1 ^mutant fungal host cell can be partially deleted, thus leaving only a portion of the CRZ1 coding sequence in the chromosomal locus where CRZ1 would naturally occur (eg, here The CRZ1 zinc finger domain may be partially or completely deleted); as a result, CRZ1 coding sequences will not occur at all in the chromosomal locus where CRZ1 would naturally occur (eg, Where CRZ1 is completely deleted and replaced with other polynucleotides such as auxotrophic markers); can be disrupted, resulting in insertion of heterologous sequences within the CRZ1 chromosomal gene; one or more within the chromosomal gene The CRZ1 can be mutated in cells compared to cells not so mutated (eg, For example, mutations that partially or completely inactivate therein may be mutated to lower CRZ1 expression levels or activity in CRZ1 zinc finger domains); Any form can be partially or completely inactivated. Alternatively, the regulatory region of the endogenous CRZ1 gene can be partially or completely deleted, destroyed or mutated so that a significant amount of functional CRZ1 polypeptide is not expressed in the cell. Furthermore, CRZ1 expression is reduced or eliminated in some way, for example by interference of expression using antisense CRZ1 molecules, SiRNA CRZ1 molecules, or by enhanced CRZ1 proteolysis, or by chemical inhibition using small molecule inhibitors. obtain. The isolated crz1 ^mutant fungal host cell is part of the present invention.

The scope of the present invention includes isolated crz1 ^mutant fungal host cells that are “viable” at high temperatures and that are more robust during fermentation, as well as the use of such cells as described herein. The viability of isolated fungal host cells in a cell culture in a bioreactor / fermenter environment at an elevated temperature such as 32 ° C., in one embodiment of the invention, measures cell lysis in the cell culture. Is determined by crz1 ^mutant fungal host cell lysis is assessed in one embodiment of the invention microscopically or by measuring the double stranded DNA content of the culture. Microscopic evaluation is performed by scoring the amount of cell debris observed in the culture. Cell debris in the culture medium is the result of cell lysis and is therefore a marker for cell lysis and a technique for measuring cell viability in the culture medium. A score of 1, 2, 3, 4 or 5 is given; 5 represents maximum lysis, ie greater than 90% cell lysis. The crz1 ^mutant fungal host cells showed a score of less than 5 for lysis for 90 to 110 hours after induction at 32 ° C. Cultures containing crz1 ^mutant fungal host cells induced at 32 ° C. had less than 30 milligrams / milliliter of double-stranded DNA for 90 to 110 hours. When the cells lyse, the double stranded DNA is released into the medium; thus the double stranded DNA content of the culture is a marker for cell lysis and is used to determine the viability of the cells in the culture It is a technique. Double-stranded DNA is a fluorescent dye (for example, indole derivative dyes such as bisbenzimidazole, Hoechst 33342, Hoechst 33258, or 49,6-diamidino bound to the double-stranded DNA in the culture medium by affinity for the double-stranded DNA. -2-phenylindole; phenanthridinium dyes such as ethidium bromide or propidium iodide; cyanine dyes such as PicoGreen; YOYO-1 iodide; SYBR Green I or SYBR Gold; (6): 585-599 (see 2001)) and may be measured using any of several methods known in the art. The content of double-stranded DNA in the culture can then be determined on this basis. As a result, cells in culture with a microscopic lysis score of less than 5 and / or a double-stranded DNA content of 30 milligrams / milliliter or less are considered “viable”.

  Isolated fungal host cells that are viable for about 90 to about 110 hours after induction at 32 ° C. are referred to herein as a “temperature-resistant” or “temperature-resistant” phenotype. It can be expressed as

The present invention includes a heterologous polynucleotide encoding a heterologous polypeptide (eg, a reporter or immunoglobulin heavy and / or light chain), wherein the heterologous polynucleotide may be operably linked to a promoter; and Methods of use thereof include, for example, methods for expressing the heterologous polypeptide in the fungal host cell. For example, the invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into a crz1 ^mutant fungal host cell, and (ii) crz1 ^mutant fungus under conditions favorable for expression of the heterologous polypeptide in the cell. Culturing the host cell, optionally, (iii) isolating the heterologous polypeptide from the crz1 ^mutant fungal host cell, optionally with one or more additional changes (eg, an endogenous gene) And / or a mutation to the expression of one or more other genes; eg, as described herein, eg, a mutation to produce a glycosylation modification of the expressed polypeptide) in a crz1 ^mutant fungal host cell (eg, Pichia) Include methods for producing a plurality of heterologous polypeptides.

In one embodiment of the invention, the crz1 ^mutant fungal host cell also comprises a mutation in ATT1. In one embodiment of the invention, the crz1 ^mutant fungal host cell does not contain a mutation in ATT1, eg, endogenous ATT1 is wild type (eg, the cell contains a wild type, functional ATT1 polypeptide), The cell and its use are part of the invention, as described herein.

  The isolated fungal host cell of the present invention is a cell belonging to the fungal kingdom. For example, in one embodiment of the present invention, the fungal host cell is any yeast such as budding yeast and / or fission yeast. In one embodiment of the present invention, the host cell is any methanol-assimilating yeast. Methanol-utilizing yeast is a small group of yeast species that can utilize methanol as the sole carbon and energy source. Methanol-assimilating yeasts include Pichia pastoris, Pichia angsta (Hansenula polymorpha), Pichia methanoli and C. It is. In one embodiment of the invention, the host cell can be any Pichia cell, such as Pichia pastoris, Pichia angusta (Hansenula polymorpha), Pichia finlanda, Pichia finlanda. (Pichia finlandica), Pichia trehalophila, Pichia coclamae, Pichia membranaefaciens, Pichia mitita e Lindneri (Pichia Lindner) )), Pichia puntiae, Pichia thermotolerans, Pichia ilicia p, Pichia pichia, Pichia pichia ) Or Pichia methanolica, etc .; Saccharomyces cerevisiae, Saccharomyces sp., Hansenula velocoli morphs p.), Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus nigers, Aspergillus zes Trichoderma reesei, Chrysosporium lucnowense, Fusarium sp., Fusarium grameneum, and Fusarium venenum It is selected from the group consisting of Neurospora crassa (Neurospora crassa). In one particular embodiment of the invention, the isolated fungal host cell is as described above except that the term excludes Saccharomyces cerevisiae.

In one embodiment of the invention, the isolated fungal host cell is glycoengineered. In one embodiment of the invention, the cell has been genetically modified to produce a glycoprotein, wherein N- or O-linked glycosylation is, for example, OCH1, ALG3, PNO1 and / or BMT1, BMT2. A gene involved in N-glycosylation such as BMT3, MBT4, or by inactivation or deletion of genes involved in O-glycosylation such as PMT1, PMT2 and / or PMT4, or GnTI, GnTII, GalT and / or SialT Is modified from its native form by heterologous expression of glycosyltransferases such as, or glycosidases such as MNSI and / or MNSII. For example, in one embodiment of the invention, the glycoengineered isolated fungal host cell has the following properties:
(I) where one or more endogenous beta-mannosyltransferase genes are mutated, disrupted, truncated or partially or completely deleted;
(Ii) comprising a polynucleotide encoding an alpha-1,2 mannosidase enzyme;
(Iii) where one or more endogenous phosphomannosyltransferases are mutated, disrupted, truncated or partially or completely deleted;
(Iv) contains a single subunit oligosaccharyl transferase (eg, Leishmania sp. STT3D);
(V) where endogenous dolichol phosphate mannose-dependent alpha-1,3-mannosyltransferase (eg ALG3) has been mutated, disrupted, truncated or partially or completely deleted;
(Vi) comprises a polynucleotide encoding endomannosidase;
(Vii) one or more encoding a bifunctional UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase, N-acetylneuraminic acid-9-phosphate synthase or CMP-sialic acid synthase Contains a polynucleotide;
(Viii) where the endogenous ATT1 gene is mutated, disrupted, truncated or partially or completely deleted;
(Ix) where endogenous alpha-1,6-mannosyltransferase (eg, OCH1) is mutated, disrupted, truncated or partially or completely deleted;
(X) comprising a polynucleotide encoding a galactosyltransferase;
(Xi) comprising a polynucleotide encoding a sugar nucleotide transporter;
(Xii) comprising a polynucleotide encoding a sialyltransferase;
(Xiii) comprising a polynucleotide encoding acetylglucosaminyltransferase and / or (xiv) where one or more endogenous proteases (eg PEP4 and PRB1) are still mutated, disrupted, truncated or partially Or one or more of which are completely deleted. As used herein, mutation of CRZ1 in a sugar engineered isolated fungal host cell reverses temperature sensitivity, enhances cell robustness in culture, and reverses poor productivity, ie, in the cell Has been shown to at least partially enhance the productivity of heterologous polypeptides such as immunoglobulins. Such sugar engineered isolated fungal host cells are part of the present invention. Methods for reversing the temperature sensitivity of glycoengineered isolated fungal host cells by mutating CRZ1 or otherwise reducing the expression of CRZ1 polypeptide and / or enhancing cell robustness during fermentation A method for this is also part of the invention.

  As used herein, the terms “N-glycan” and “glycoform” are used interchangeably, and an asparagine-N-acetylglucosamine bond to an N-linked oligosaccharide, eg, an asparagine residue of a polypeptide. Represents an N-linked oligosaccharide bound by N-linked glycoproteins contain N-acetylglucosamine residues that bind to the amide nitrogen of asparagine residues in the protein. The main sugars found in glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (eg, N-acetyl-neuraminic acid (NANA)) is there.

N-glycans represent the common pentasaccharide core Man ₃ GlcNAc ₂ (“Man” represents mannose; “Glc” represents glucose; and “NAc” represents N-acetyl; GlcNAc represents N-acetylglucosamine ). N-glycans are peripheral sugars (eg, GlcNAc, galactose) added to the Man ₃ GlcNAc ₂ (“Man ₃ ”) core structure, also referred to as “trimannose core”, “pentose core” or “pouch mannose core”. , Fucose and sialic acid), depending on the number of branches (antennas). N-glycans are classified according to their branched constituents (eg, high mannose, complex or hybrid). “High mannose” type N-glycans have five or more mannose residues. “Complex” type N-glycans typically have at least one GlcNAc bound to the 1,3 mannose arm of the “trimannose” core and at least one GlcNAc bound to the 1,6 mannose arm of the core. Complex N-glycans are also optionally modified with sialic acid or derivatives (eg, “NANA” or “NeuAc”, where “Neu” represents neuraminic acid and “Ac” represents acetyl) It also has galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residues. Complex N-glycans may also have intrachain substituents including “bifurcated” GlcNAc and core fucose (“Fuc”). Complex N-glycans can also have multiple antennas on the “Trimannoscore” and are often referred to as “multi-antenna glycans”. “Hybrid” N-glycans have at least one GlcNAc on the end of the 1,3 mannose arm of the trimannose core and zero or more mannose on the 1,6 mannose arm of the trimannose core. Various N-glycans are also referred to as “glycoforms”. “Peptide: N-glycanase (PNGase)” or “glycanase” refers to peptide N-glycosidase F (EC 3.2.2.18).

In one embodiment of the invention, an isolated crz1 ^mutant fungal host cell of a Pichia cell or the like (eg, Pichia pastoris) is α-1, which has a signal peptide that directs secretion. It is genetically modified to contain a nucleic acid encoding 2-mannosidase. For example, in one embodiment of the present invention, the crz1 ^mutant host cell has an exogenous α-1,2-mannosidase enzyme having an optimal pH between 5.1 and 8.0, preferably between 5.9 and 7.5. To be expressed. In one embodiment of the invention, the exogenous enzyme targets the endoplasmic reticulum or Golgi apparatus of the host cell, where the enzyme trims N-glycans such as Man ₈ GlcNAc ₂ to yield Man ₅ GlcNAc ₂ . See U.S. Patent No. 7,029,872. The present invention comprises (i) introducing a polynucleotide encoding a heterologous polypeptide into the crz1 ^mutant , α-1,2-mannosidase ⁺ host cell, and (ii) expressing the heterologous polypeptide in the cell. A method for producing one or more heterologous polypeptides comprising culturing the host cells under favorable conditions and, optionally, (iii) isolating the heterologous polypeptide from the host cell. including. The present invention also includes an N-glycan structure comprising a Man ₅ GlcNAc ₂ glycoform in a crz1 ^mutant fungal host cell that does not exhibit alpha-1,6 mannosyltransferase (eg, OCH1) activity for N-glycans on glycoproteins also encompasses a method for producing heterologous recombinant glycoproteins including, the method crz1 ^mutant och1 ^- fungal host cell, a polynucleotide encoding a heterologous recombinant glycoproteins, and in the ER or Golgi apparatus of the host cell Introducing a polynucleotide encoding an alpha-1,2 mannosidase enzyme selected to have optimal activity, said enzyme comprising (a) a pH between 5.1 and 8.0 in said ER or Golgi apparatus Alpha-1,2 having optimal activity (B) fused to a target signal peptide of a cell not normally associated with the catalytic domain selected to target the mannosidase enzyme to the ER or Golgi apparatus of the host cell; and heterologous recombination N-glycan bound to the fungal host cell under conditions favorable for expression of the glycoprotein, whereby the heterologous recombinant glycoprotein is expressed and passed through the ER or Golgi body of the host cell More than 30 mol% of the structure has a Man ₅ GlcNAc ₂ glycoform that can serve as a substrate for GlcNAc transferase I in vivo.

An isolated crz1 ^mutant fungal host cell of the present invention of a Pichia host cell or the like (eg, Pichia pastoris) is, in one embodiment of the present invention, a β-mannosyltransferase gene (eg, BMT1, BMT2, BMT3 and / or BMT4) (see US Pat. No. 7,465,577) by deleting or destroying one or more or using interfering RNA, antisense RNA, etc. It has been genetically modified to eliminate glycoproteins with alpha-mannosidase resistant N-glycans by inhibiting translation of RNA encoding one or more mannosyltransferases. The scope of the present invention, (i) said heterologous polypeptide encoding polynucleotide the ^{crz1 mutant, β-} mannosyltransferase ^{- (e.g., bmt1 -, bmt2 -, bmt3} - and / or Bmt4 ^-) introduced into the host cell And (ii) culturing the host cell under conditions favorable for expression of the heterologous polypeptide in the cell, and optionally (iii) isolating the heterologous polypeptide from the host cell A method for producing one or more heterologous polypeptides.

The isolated crz1 ^mutant fungal host cells of the invention (eg, Pichia, eg, Pichia pastoris) also use one or more phosphomannosyltransferases using interfering RNA, antisense RNA, etc. Phosphomannosyltransferase genes PNO1 and MNN4B (see, eg, US Pat. No. 7,198,921 and), which may comprise deleting or destroying, or thus inhibiting translation of RNA encoding one or more of the phosphomannosyltransferases Also included are such cells that have been genetically modified to remove glycoproteins having phosphomannose residues by deleting or destroying one or both of (see 7,259,007). In one embodiment of the invention, the fungal host cell produces a glycoprotein primarily comprising an N-glycan selected from the group consisting of complex N-glycans, mixed N-glycans and high mannose N-glycans; Here, the complex type N-glycan is, in one embodiment of the present invention, Man ₃ GlcNAc ₂ , GlcNAC _(1-4) Man ₃ GlcNAc ₂ , NANA _(1-4) GlcNAc _(1-4) Man ₃ GlcNAc ₂ and NANA _(1-4) Gal _(1-4) Man ₃ GlcNAc ₂ is selected from the group consisting of; N-glycan in one embodiment of the present invention is Man ₅ GlcNAc ₂ , GlcNAcMan ₅ GlcNAc ₂ , GalGlcNAcMan ₅ GlcNAc ₂ and NANAGalGlcN is selected from the group consisting of cman ₅ GlcNAc _2; and high-mannose type N- glycans, in one embodiment of the present _{invention, Man 6 GlcNAc 2, Man 7} GlcNAc 2, Man 8 group consisting of GlcNAc ₂ and _Man 9 GlcNAc ₂ More selected. The scope of the present invention, (i) said heterologous polypeptide the polynucleotides encoding ^{Crz1 Mutant,} phosphoribosyl mannosyltransferase ^- (e.g., PNO1 ^- and / or MNN4B ^-) is introduced into a host cell, and (ii) One or more comprising culturing the host cell under conditions favorable for expression of the heterologous polypeptide in the cell, and optionally (iii) isolating the heterologous polypeptide from the host cell. A method for producing a heterologous polypeptide of is included.

An isolated crz1 ^mutant fungal host cell, such as a Pichia host cell of the invention (eg, Pichia pastoris), includes a single subunit oligosaccharide transferase of Leishmania sp. Included are isolated crz1 ^mutant fungal host cells that have been genetically modified to contain nucleic acids such as those described in WO2011 / 06389 that encode STT3A, STT3B, STT3C, STT3D, or combinations thereof. The scope of the present invention includes: (i) a polynucleotide encoding the heterologous polypeptide, wherein the crz1 ^mutant , Leishmania STT3A ⁺ , Leishmania STT3B ⁺ , Leishmania STT3C ⁺ And / or introduction into a Leishmania STT3D ⁺ host cell, and (ii) culturing the host cell under conditions favorable for expression of the heterologous polypeptide in the cell, and optionally , (Iii) a method for producing one or more heterologous polypeptides comprising isolating the heterologous polypeptide from the host cell.

The isolated crz1 ^mutant fungal host cells of the invention (eg, Pichia pastoris) also include dolichol phosphate mannose-dependent alpha (1- 3) Also included are isolated crz1 ^mutant fungal host cells that have been genetically modified to remove nucleic acids encoding mannosyltransferases, eg, ALG3. The scope of the present invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , alg3 ^- host cell, and (ii) conditions favorable for expression of the heterologous polypeptide in the cell. A method for producing one or more heterologous polypeptides, comprising culturing the host cell with, and optionally (iii) isolating the heterologous polypeptide from the host cell. .

An isolated crz1 ^mutant fungal host cell of the invention, eg, a poly having endomannosidase activity (eg, human (eg, human liver), rat or mouse endomannosidase) that targets the endoplasmic reticulum compartment within the host cell. Pichia cells that express peptides (eg, Pichia pastoris) and the like are part of the present invention. The scope of the present invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , endomannosidase ⁺ host cell, and (ii) favorable conditions for expression of the heterologous polypeptide in the cell. Culturing the host cell under, and optionally (iii) isolating the heterologous polypeptide from the host cell, comprising a method for producing one or more heterologous polypeptides It is.

An isolated crz1 ^mutant fungal host cell, such as a Pichia cell of the invention (eg, Pichia pastoris), etc., in one embodiment of the invention, is a recombinant sialylated sugar in the host cell. For example, wherein the host cell comprises (a) a bifunctional UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase, N-acetylneuramin Transforming one or more polynucleotides encoding acid-9-phosphate synthase and CMP-sialic acid synthase into a crz1 ^mutant fungal host cell; (b) a polynucleotide encoding a CMP-sialic acid transporter; Transforming a host cell; and (c For example, S. Transforming the host cell with a polynucleotide molecule encoding a 2,6-sialyltransferase catalytic domain fused to a cellular targeting signal peptide encoded by nucleotides 1-108 of S. cerevisiae Mnn2. Wherein the host cell is selected or modified to produce a recombinant glycoprotein comprising a glycoform selected from the group consisting of Gal _(1-4) GlcNAc _(1-4) Man ₃ GlcNAc ₂ Including a glycoform selected from the group consisting of NANA _(1-4) Gal _(1-4) GlcNAc _(1-4) Man ₃ GlcNAc glycoforms upon passage of the recombinant glycoprotein via the secretory pathway of Recombinant sialylated glycoprotein is produced The scope of the present invention includes: (i) a polynucleotide encoding said heterologous polypeptide, said crz1 ^mutant , bifunctional UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase ⁺ , N- Acetylneuraminic acid-9-phosphate synthase ⁺ , CMP-sialic acid synthase ⁺ , CMP-sialic acid transporter ⁺ , 2,6-sialyltransferase ⁺ introducing into a host cell, and (ii) the cell in the cell One or more heterologous polypeptides comprising culturing the host cell under conditions favorable for expression of the heterologous polypeptide, and optionally (iii) isolating the heterologous polypeptide from the host cell A method for producing is included.

In addition, isolated crz1 ^mutant fungal host cells of the invention, such as Pichia cells (eg, Pichia pastoris), etc., in one embodiment of the invention, may contain, for example, terminal galactose on the glycoprotein. Modified to produce a galactosylated protein having residues and substantially lacking fucose and sialic acid residues. In one embodiment of the invention, the isolated crz1 ^mutant fungal host cell comprises an isolated nucleic acid molecule encoding β-galactosyltransferase activity and UDP-galactose transport activity, UDP-galactose C4 epimerase activity, galactokinase-1 or galactose-1 -Containing at least one nucleotide encoding uridyl phosphate transferase, for example, wherein the host cell has been genetically modified to produce an N-linked oligosaccharide having a terminal GlcNAc residue; and A polynucleotide encoding a fusion protein that transfers a galactose residue from a UDP-galactose to a terminal GlcNAc residue of an N-linked oligosaccharide branch of the N-glycan of a glycoprotein in a host cell, wherein N -Join The ligosugar branch is selected from the group consisting of GlcNAcβ1,2-Manα1, GlcNAcβ1,4-Manα1,3, GlcNAcβ1,2-Manα1,6, GlcNAcβ1,4-Manα1,6 and GlcNAcβ1,6-Manα1,6; And the host cell has reduced or depleted dolylylphosphate mannose: Man ₅ GlcNAc ₂ -PP-Drykyl α-1,3 mannosyltransferase activity, wherein the host cell is a sugar having one or more galactose residues Produce protein. The scope of the present invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the host cell, and (ii) allowing the host cell to Included is a method for producing one or more heterologous polypeptides comprising culturing, and optionally (iii) isolating the heterologous polypeptide from the host cell.

In one embodiment of the invention, an isolated crz1 ^mutant fungal host cell of the invention, such as a Pichia cell (eg, Pichia pastoris), etc. lacks a functional OCH1 protein, for example, Here, endogenous OCH1 is mutated. The scope of the invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , och1 ^- host cell, and (ii) conditions favorable for expression of the heterologous polypeptide in the cell. A method for producing one or more heterologous polypeptides, comprising culturing the host cell with, and optionally (iii) isolating the heterologous polypeptide from the host cell. .

An isolated crz1 ^mutant fungal host cell of the invention, eg, a Pichia cell (eg, Pichia pastoris expressing a galactosyltransferase, eg, alpha 1,3-galactosyltransferase or beta 1,4-galactosyltransferase, eg, Pichia pastoris )) Etc. are part of the present invention. The scope of the present invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , galactosyltransferase ⁺ host cell, and (ii) favorable conditions for expression of the heterologous polypeptide in the cell. Culturing the host cell under, and optionally (iii) isolating the heterologous polypeptide from the host cell, comprising a method for producing one or more heterologous polypeptides It is.

Isolated crz1 ^mutant fungal host cells of the invention, Pichia cells (eg, Pichia pastoris) that express a sugar nucleotide transporter, and the like are part of the invention. The scope of the present invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , sugar nucleotide transporter ⁺ host cell, and (ii) expressing the heterologous polypeptide in the cell. A method for producing one or more heterologous polypeptides, comprising culturing the host cells under favorable conditions, and optionally (iii) isolating the heterologous polypeptide from the host cells. Is included.

Isolated crz1 ^mutant fungal host cells of the present invention, such as Pichia cells expressing sialyltransferase (eg, Pichia pastoris), etc. are part of the present invention. The scope of the present invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , sialyltransferase ⁺ host cell, and (ii) favorable conditions for expression of the heterologous polypeptide in the cell. Culturing the host cell under, and optionally (iii) isolating the heterologous polypeptide from the host cell, comprising a method for producing one or more heterologous polypeptides It is.

An isolated crz1 ^mutant fungal host cell of the invention, such as an acetylglucosaminyl transferase, such as a Pichia cell (eg, Pichia pastoris) expressing GNT1 or GNT2 or GNT4, etc. Part of the invention. The scope of the invention includes (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant , acetylglucosaminyltransferase ⁺ host cell, and (ii) expression of the heterologous polypeptide in the cell. For producing one or more heterologous polypeptides, comprising culturing the host cells under conditions favorable to, and optionally (iii) isolating the heterologous polypeptide from the host cells. Methods are included.

  As used herein, the term “essentially free of” refers to a glycoprotein component as related to the absence of a particular sugar residue, fucose or galactose, etc. on the glycoprotein. Used to indicate a substantial lack of N-glycans containing residues. When expressed in terms of purity, it is substantially free means that the amount of N-glycan structure containing the sugar residue does not exceed 10%, preferably less than 5%, more preferably less than 1% Most preferably less than 0.5%, where the percentages are by weight or mole percent.

  As used herein, when a glycoprotein composition “lacks” or “is lacking” a particular sugar residue, such as fucose or galactose, a detectable amount of the sugar residue The group is not present on the N-glycan structure. For example, in one embodiment of the invention, a glycoprotein composition produced by a host cell of the invention can be “lac fucose” because the cell has fucosylated N-glycan structure. This is because it does not have the enzyme required for production. Thus, the term “substantially free of fucose” encompasses the term “lacking fucose”. However, even if the composition originally contained a fucosylated N-glycan structure, or even a small but detectable amount of fucosylated N-glycan structure as described above, the composition It may be “substantially free of fucose”.

  The scope of the present invention is that only one endogenous chromosomal CRZ1 gene is mutated, disrupted, truncated or partially or completely deleted, and the other endogenous chromosomal CRZ1 gene is mutated, disrupted, Includes diploid isolated fungal host cells that are not truncated or partially or completely deleted and that encode a functional CRZ1 polypeptide. For example, homodiploids that lack a functional CRZ1 polypeptide because the endogenous chromosomal copies of both CRZ1 genes are mutated, disrupted, truncated, or partially or completely deleted are also claimed in the present invention. Is part of.

Protein expression Within the scope of the present invention is (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 ^mutant host cell (eg, as described herein), and (ii) the cell in the cell. Culturing the host cell under conditions favorable for expression of the heterologous polypeptide, as long as the cell is viable, and, optionally, (iii) isolating the heterologous polypeptide from the host cell A method for producing one or more heterologous polypeptides. Methods for expressing heterologous polypeptides in fungal host cells are generally known and common in the art.

  The invention includes any fungal host cell suspended in a liquid culture medium as described herein. Any lysate of isolated fungal host cells described herein is also within the scope of the invention.

The culture conditions used for the fungal host cell expression system can vary depending on the individual conditions at hand. In one embodiment of the invention, the fungal host cell is a liquid in a shake flask or fermenter (eg, 1 L, 2 L, 5 L, 10 L, 20 L, 30 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 10,000 L). Can be grown in media. Various growth media can be used to culture the fungal host cells. In one embodiment of the invention, the medium is at a pH between pH 3 and 7 (eg, 3, 4, 5, 6 or 7); in one embodiment of the invention, the pH is a base such as ammonium hydroxide. Is raised by. In one embodiment of the invention, the temperature is maintained at about 24 ° C. or 26 ° C. or 28 ° C. or 30 ° C. or 32 ° C. or 34 ° C. In one embodiment of the invention, the dissolved oxygen in the growth medium is maintained at about 20% or 30%. In one embodiment of the present invention, the growth medium contains yeast nitrogen source basal medium (eg, ammonium sulfate added; essential amino acid added or not), peptone and / or yeast extract. Various supplements such as biotin, dextrose, methanol, glycerin, casamino acid, L-arginine hydrochloride, ammonium ions (eg, ammonium phosphate), etc. can be added to the growth medium. In one embodiment of the present invention, the growth medium is a minimal medium containing a yeast nitrogen source basal medium, a carbon source such as water, dextrose, methanol or glycerin, biotin and histidine. In one embodiment of the present invention, the cell culture medium comprises copper, iodine, manganese, molybdenum, boron, cobalt, zinc, iron, biotin and / or sulfur, such as CuSO ₄ , NaI, MnSO ₄ , Na ₂ MoO ₄ , H _3. Trace minerals / nutrients such as BO ₃ , CoCl ₂ , ZnCl ₂ , FeSO ₄ , biotin and / or H ₂ SO ₄ are included. In one embodiment of the present invention, the cell culture medium includes an antifoaming agent (eg, silicon).

The present invention relates to an isolated fungal crz1 ^mutant host cell (eg, Pichia, eg, Pichia pastoris, etc.) operably linked to, eg, a promoter, eg, a methanol-inducible promoter. Introducing a heterologous polynucleotide encoding said polypeptide, and (i) in a batch phase where the cells are grown with a non-fermentable carbon source such as glycerin (eg glycerin batch phase), eg non-fermenting Until the carbon source is depleted;
(Ii) in an added batch phase (eg, glycerin added batch phase) where an additional non-fermentable carbon source (eg, glycerin) is fed, eg, at a growth limiting rate; and (iii) the cells are methanol and optional Optionally, producing a heterologous polypeptide (eg, an immunoglobulin chain or antibody or antibody-binding fragment thereof) comprising culturing the host cell in a methanol-added batch phase that is grown in the presence of additional glycerin To include a method for

  In one embodiment of the invention, the methanol concentration during the methanol addition batch phase is about 2 grams / liter methanol to about 5 grams / liter methanol (eg, 2, 2.5, 3, 3.5, 4, 4.5). Or 5).

In one embodiment of the invention, prior to the batch phase, the initial seed culture is grown to a high density (eg, OD ₆₀₀ of about 2 or greater) and then the seed culture proliferating cells are transferred to the initial batch phase culture. Used to inoculate.

  In one embodiment of the invention, host cells are grown in a transformation phase in which cells are grown in the presence of about 2 ml of methanol per liter of culture broth after the addition batch phase and before the methanol addition batch phase. For example, the cells can be grown in the conversion phase until the methanol concentration is about zero.

  A heterologous polypeptide isolated from a fungal host cell is purified in one embodiment of the invention. If the heterologous polypeptide is secreted from the fungal host cell into a liquid growth medium, the polypeptide can be purified by a process that includes removal of the fungal host cell from the growth medium. Removal of the cells from the medium can be performed by centrifugation, the cells are discarded and the liquid medium supernatant is pooled. If the heterologous polypeptide is not secreted, the liquid medium is discarded after separation from the fungal host cells and the cells are retained. The fungal host cell can then be lysed to produce a crude cell lysate from which the heterologous polypeptide is further purified.

  Purification of the heterologous polypeptide is performed in one embodiment of the invention by chromatography, eg, column chromatography. Chromatographic purification can include the use of ion exchange, such as anion exchange and / or cation exchange, protein A chromatography, size exclusion chromatography and / or hydrophobic interaction chromatography. Purification includes virus inactivation, precipitation and / or lyophilization of the composition comprising the polypeptide.

[Example]
This section is intended to describe the invention in greater detail and should not be construed as further limiting the invention. Any composition or method set forth herein forms part of the present invention.

[Example 1]
Identification of CRZ1 mutation.

Experimental Methods UV-induced mutation, fed-batch culture, IgG purification, N-glycan characterization (characterization), and all other analytical assays were performed as previously described (Barnard et al. 2010; Jiang et al. 2011; Potgieter et al. 2009; Winston F2008). Unless otherwise indicated, the 1 L bioreactor fermentation run was planned to end 100-120 hours after MeOH introduction. However, if excessive cell lysis was observed, the fermentation process was completed prematurely. Cell lysis was quantified by microscopic examination or by measuring the amount of nuclear DNA released into the supernatant (Barnard, 2010). Excess cell lysis was defined by greater than 90% lysed cells by microscopic examination or a DNA concentration greater than 30 milligrams / ml in the supernatant as measured by the Picogreen assay.

Results and Discussion A thermostable mutant exhibiting substantial enhancement of fermentation robustness.

  To identify Pichia host strains with enhanced fermentation robustness, we have the theory that specific second site mutations that suppress temperature-sensitive defects can also compensate for cellular robustness deficiencies Under the rationale, temperature sensitive sugar engineered strains (YGLY12905, YGLY22835 and YGLY27890) were UV-induced mutated to select thermostable mutants. After confirming their thermotolerant phenotype, these mutants were fermented using a standard MeOH addition batch process in a 1 L DasGip bioreactor. After an extensive fermentation screening campaign, the inventors identified nine mutant strains that showed a significant increase in cellular robustness during the fermentation process. As shown in FIG. 1, the culture process for the non-mutagenized control strain had to be terminated due to excessive cell lysis at about 48 hours of induction at 32 ° C. In contrast, all of the mutant strains showed a marked improvement in fermentation robustness. YGLY29010, YGLY29030, YGLY29031, and YGLY29012 were able to ferment for about 90 hours;

  Protein productivity of thermostable mutants and quality assessment of N-glycans.

  Five thermostable mutant strains (YGLY29010, YGLY29030, YGLY29031, YGLY29042 and YGLY29012) were derived from YGLY27890 expressing human Fc fragment (FIG. 2). In order to assess how these thermostable mutations affected Fc productivity and N-glycan quality, we purified the Fc fragment from the 1 L bioreactor at 32 ° C. The valence was quantified (FIG. 1), and the profiles (characteristics) of N-glycans of four thermostable mutant strains and the non-mutagenized parent strain YGLY27890 were analyzed (FIG. 3). Four mutant strains (YGLY29010, YGLY29030, YGLY29031 and YGLY29042) showed a substantial increase in product titers compared to the parental control: YGLY29042 and YGLY29010 were actually about 4-5 times more The Fc product was secreted. In contrast, the product titer from YGLY29012 was only about 50% of that of the control strain. For N-glycans derived from Fc products, we did not observe any significant changes in N-glycan profiles (Table 3). Similar to the control strain YGLY27890 (A2 83% and A1 4%) all 5 mutants had A2 levels ranging from 77 to 84% and 4 to 7% with high levels of terminal sialic acid Up to the A1 level, the FcN-glycan could be effectively modified. Taken together, these results indicate that the UV-induced mutations acquired by YGLY29010, YGLY29030, YGLY290301 and YGLY29042 do not adversely affect the ability to produce non-homologously expressed human Fc fragments. Revealed that it did not cause significant degradation in the quality of N-glycans.

  Genomic sequencing to identify causal mutations responsible for enhanced thermostability and fermentation robustness.

  To better understand the molecular mechanisms involved in maintaining cellular robustness during fermentation, we have determined that these nine thermostable mutants, as well as two non-mutagen empty host strains YGLY22812 and YGLY22835. The genome of was sequenced. After a genome-wide comparison between the mutant strain and the non-mutagenized strain, the inventors found that 1 to 7 non-synonymous mutations (“+” in FIG. 4) in each of these 9 mutant strains. Identified). Three mutants, YGLY29010, YGLY29031 and YGLY29042, contained a single mutation in one gene, Pp02g02120, which showed a high level of sequence homology to the Saccharomyces cerevisiae CRZ1 gene. Surprisingly, distinct mutations in the same PpCRZ1 gene were also detected in YGLY28997, YGLY28998, YGLY17159, YGLY28999, YGLY29030 and YGLY29012. ScCRZ1 is a calcineurin-responsive zinc finger transcription factor involved in stress response. The zinc finger domains are very well conserved between PpCRZ1 and ScCRZ1, both located at the c-terminus (see sequence alignment above). As illustrated in FIG. 5, the four nonsense, stop codon mutations found from the thermostable mutant were located upstream of the zinc finger, so that all of the truncated PpCRZ1 without the zinc finger domain was truncated. A fragment was generated. The remaining five mutants contained missense, amino acid substitution mutations, all of which were located in the zinc finger domain, and four of them were clustered within 25 nucleotides. The discovery that nine independently isolated thermostable mutants contained different nonsense or missense mutations within the PpCRZ1 gene indicates that these CRZ1 mutations are thermostable and enhanced fermentation robust It strongly suggested that it was the cause of phenotype. In addition, two of the mutant strains (yGLY17159 and yGLY28998) also had additional mutations in PpATT1, a gene previously shown to play an important role in thermostability and cellular robustness. . The fact that both yGLY17159 and yGLY28998 were one of the most robust mutant strains isolated was that the combination of ATT1 and CRZ1 mutations was thermostable and fermentative robust in the Pichia strain. It has been suggested that it may have an additive or synergistic effect on the enhancement of.

  PpCRZ1 sequence.

（配列番号２）
２．ｙＧＬＹ２８９９７から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｌ３３→ＳＴＯＰ、突然変異したヌクレオチドは太字体である）

（突然変異ｔ９８ａを含む配列番号２）
３．ｙＧＬＹ２９０１２から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｑ２１４→ＳＴＯＰ、突然変異したヌクレオチドは太字体である）

（突然変異ｃ６４０ｔを含む配列番号２）
４．ｙＧＬＹ２９０１０から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｌ２９４→ＳＴＯＰ、突然変異したヌクレオチドは太字体である）

（突然変異ｔ８８１ｇを含む配列番号２）
５．ｙＧＬＹ２９０４２から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｓ２９８→ＳＴＯＰ、突然変異したヌクレオチドは太字体である）

（突然変異ｃ８９３ａを含む配列番号２）
６．ｙＧＬＹ２８９９８から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｅ４０３→Ｇ、突然変異したヌクレオチドは太字体である）

（突然変異ａ１２０８ｇを含む配列番号２）
７．ｙＧＬＹ１７１５９から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｆ４０６→Ｓ、突然変異したヌクレオチドは太字体である）

（突然変異ｔ１２１７ｃを含む配列番号２）
８．ｙＧＬＹ２８９９９から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｆ４０６→Ｌ、突然変異したヌクレオチドは太字体である）

（突然変異ｔ１２１６ｃを含む配列番号２）
９．ｙＧＬＹ２９０３０から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｃ４１１→Ｆ、突然変異したヌクレオチドは太字体である）

（突然変異ｇ１２３２ｔを含む配列番号２）
１０．ｙＧＬＹ２９０３１から単離されたピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）突然変異株ＣＲＺ１（コードする突然変異：Ｋ４６９→Ｎ、突然変異したヌクレオチドは太字体である）

（突然変異ａ１４０７ｃを含む配列番号２）
［実施例２］
指向的菌株改変による表現型の確認
本明細書で述べる通り、突然変異株それぞれの同じＣＲＺ１遺伝子における独立した突然変異は、この転写因子の不活性化が観察された耐熱性および発酵ロバスト性表現型の原因であることを強く示唆した。この結論を確認するために図８に示す通り、５‐ＦＯＡ対抗選択によりＹＧＬＹ１７１０８に由来する非突然変異誘発ｕｒａ５栄養要求性ピキア属（Ｐｉｃｈｉａ）菌株であるＹＧＬＹ２１２０３において、ＣＲＺ１ ＯＲＦが完全に欠失されるか、または内在性ＣＲＺ１遺伝子がトランケート化されたバージョンと置換された。 1. Pichia pastoris CRZ1 wild type open reading frame

(SEQ ID NO: 2)
2. Pichia pastoris mutant CRZ1 isolated from yGLY28997 (coding mutation: L33 → STOP, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation t98a)
3. Pichia pastoris mutant CRZ1 isolated from yGLY29012 (coding mutation: Q214 → STOP, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation c640t)
4). Pichia pastoris mutant CRZ1 isolated from yGLY29010 (encoding mutation: L294 → STOP, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation t881g)
5. Pichia pastoris mutant CRZ1 isolated from yGLY29042 (coding mutation: S298 → STOP, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation c893a)
6). Pichia pastoris mutant CRZ1 isolated from yGLY28998 (coding mutation: E403 → G, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation a1208g)
7). Pichia pastoris mutant CRZ1 isolated from yGLY17159 (coding mutation: F406 → S, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation t1217c)
8). Pichia pastoris mutant CRZ1 isolated from yGLY28999 (coding mutation: F406 → L, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation t1216c)
9. Pichia pastoris mutant CRZ1 isolated from yGLY29030 (coding mutation: C411 → F, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation g1232t)
10. Pichia pastoris mutant CRZ1 isolated from yGLY29031 (coding mutation: K469 → N, mutated nucleotides are in bold)

(SEQ ID NO: 2 containing mutation a1407c)
[Example 2]
Confirmation of Phenotype by Directed Strain Modification As described herein, independent mutations in the same CRZ1 gene of each mutant strain are thermostable and fermentation robust phenotypes where inactivation of this transcription factor has been observed It was strongly suggested that To confirm this conclusion, as shown in FIG. 8, the CRZ1 ORF was completely deleted in YGLY21203, a non-mutagenized ura5 auxotrophic Pichia strain derived from YGLY17108 by 5-FOA counterselection. Or the endogenous CRZ1 gene was replaced with a truncated version.

Plasmid pGLY12829 (FIG. 6) was constructed by cloning a 1.5 kb genomic DNA fragment immediately upstream of the CRZ1 ORF, followed by the lacZ-URA5-lacZ URA blaster and before the ALG3 terminator, and then CRZ1 It was ligated to a 2 kb genomic DNA fragment containing the last 234 bp of the ORF and 1.7 kb of the downstream region. After SfiI digestion, this CRZ1-upstream-URA blaster-CRZ1-downstream DNA fragment was transformed into a non-mutagenized host strain (eg, YGLY17108). By homologous recombination in both the CRZ1 upstream and downstream regions, this URA blaster-cassette replaced the endogenous CRZ1 gene while deleting 85% of the coding region of CRZ1, resulting in a CRZ1 complete knockout mutant . To confirm the correct substitution of the CRZ1 ORF, the following oligonucleotides as PCR primers: GTATGCGATATAGTGTGGA (SEQ ID NO: 4, 4,15435 bp upstream of the CRZ1 start point) and TGGGGAGAAGGTACCGAAG (SEQ ID NO: 5) to confirm the 5 ′ junction of gene replacement 5, within ALG3 terminator); CACTACCGCGTACTGTGAGCC (within SEQ ID NO: 6, lacZ) and AGGATATCAAACCCCGACCAG (SEQ ID NO: 7, 7,2045 bp downstream of the CRZ1 stop codon) to confirm the 3 ′ junction of gene replacement; GACACATGCGAAATGTCTCTG (sequence cancer No. 8, 120 bp below CRZ1 stop codon to confirm omission Flow)) and TTGAGTTGGCAGCTCTCTCAG (Sequence Cancer No. 9, within the CRZ1 ORF, 1075 bp after the start) were used to perform a polymerase chain reaction (PCR) assay of genomic DNA. Plasmid pGLY12832 (FIG. 7) removes a 1.5 kb genomic DNA fragment before the ALG3 terminator accompanied by the lacZ-URA5-lacZ URA blaster (upstream region of 0.6 kb, the first 879 bp and 2 terminations of the CRZ1 ORF). Codon) and was then ligated to a 2 kb genomic DNA fragment containing the last downstream 234 bp of the CRZ1 ORF and 1.7 kb of the downstream region. Homologous recombination-mediated double crossover between the SfiI fragment of pGLY12832 (FIG. 7) and the chromosomal CRZ1 region replaced the endogenous CRZ1 ORF with a truncated version of CRZ1 having only the first 294 amino acid residues. .

  After confirming that the DNA construct correctly replaced the endogenous CRZ1 gene with the corresponding deletion or truncation, examined its ability to grow at 35 ° C on solid media and at 32 ° C in liquid media in bioreactors did. These CRZ1Δ truncated and deletion mutants may display a thermostable phenotype very similar to that observed in the original mutants isolated by UV range induction. confirmed.

  The truncated DasGip MeOH-added batch fermentation process (Hopkins et al., Then) was then used to examine whether truncated strains and deletion mutants exhibited enhanced fermentation robustness even at 32 ° C. 2011). As shown in FIG. 9, the control strain YGLY17108 showed severe lysis and was unable to survive within 65 hours of MeOH induction at 32 ° C. In contrast, strains with total deletion of the CRZ1 gene and truncation mutations showed a significant increase in fermentation robustness and successfully completed more than 130 hours of MeOH induction. These results revealed that inactivation of CRZ1 by completely or partially deleting the CRZ1 ORF is sufficient to improve the fermentation robustness of glycoengineered strains. Furthermore, the phenotypes exhibited by these directional gene replacement strains are closely similar to those exhibited by the corresponding UV-induced mutants, and mutations within the CRZ1 gene are observed in UV-induced mutants. It was explained that it was the cause of the improved heat resistance and fermentation robustness.

  Inactivation of the ATT1 gene resulted in a dramatic improvement in the fermentation robustness of the strain. Since inactivation of CRZ1 and ATT1 caused a very similar phenotype (ie, improved thermostability and fermentation robustness), the inventors have fermented strains in which the CRZ1 deletion already contains an ATT1 deletion mutation. I would like to consider whether to improve the robustness further. To achieve this goal, the inventors constructed a crz1, att1 double deletion mutant and performed its fermentation robustness by performing a standard MeOH addition batch fermentation process at 34 ° C. in a DasGip bioreactor. Was tested. Under this very stringent fermentation condition, the att1 single deletion strain YGLY29128 was viable for 75 hours, while the crz1, att1 double mutant was viable for more than 115 hours. (FIG. 10). These results show that the improvement in fermentation robustness derived from the att1 deletion and the crz1 deletion is additive, and that the crz1, att1 double mutant has a higher level of fermentation robustness than the single mutant It was clearly clarified that

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The present invention is not limited to the scope by the specific examples described herein. Indeed, the scope of the invention includes embodiments specifically set forth herein and other embodiments not specifically set forth herein; embodiments specifically set forth herein include Not necessarily intended to be exhaustive. Various modifications in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the claims.

  Patents, patent applications, literature, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated by reference in their entirety for all purposes.

Claims (14)

A functionally CRZ1 polypeptide isolated lacking expression of the gene encoding the Pichia pastoris (Pichia pastoris) host cell, wherein said Pichia pastoris (Pichia pastoris) host cell fermenter Or the isolated Pichia pastoris host cell having a thermotolerant phenotype of about 90 to about 110 hours at 32 ° C. after methanol induction in a bioreactor . The endogenous CRZ1 gene has been mutated, disrupted, partially deleted or completely deleted; or the expression of the CRZ1 polypeptide has been reduced by interfering with the transcription of the CRZ1 gene or the translation of the CRZ1 gene, or CRZ1 poly decomposition is increased peptide; or, CRZ1 activity of the polypeptide is inhibited by a chemical inhibitor, Pichia pastoris isolated according to claim 1 (Pichia pastoris) host cell. (I) Endogenous CRZ1 gene is selected from the group consisting of: L33 → STOP; Q214 → STOP; L294 → STOP; S298 → STOP; E403 → G; F406 → S; F406 → L: C411 → F; and K469 → N in contain one or more mutations are encodes a polypeptide differing from the wild-type CRZ1 polypeptide; or (ii) endogenous CRZ1 genes: a1407c; g1232t: t1216c; t1217c ; a1208g; c893a; t881g; c640t; and including one or more mutations selected from the group consisting of t98a, comprising a nucleotide sequence that differs from the wild-type CRZ1 gene ; or (iii) an endogenous CRZ1 gene that is functional C Terminal zinc fingered It differs from the wild-type CRZ1 gene in that no encoding in,
Either isolated by Pichia pastoris according to claim 1 to 2 (Pichia pastoris) host cell. (I) one or more endogenous beta-mannosyltransferase genes are mutated, disrupted, truncated or partially deleted or wholly deleted;
(Ii) comprising a polynucleotide encoding an alpha-1,2 mannosidase enzyme;
(Iii) one or more endogenous genes encoding phosphomannosyltransferase have been mutated, disrupted, truncated or partially deleted or wholly deleted;
(Iv) comprising a polynucleotide encoding a single subunit oligosaccharyltransferase;
(V) the endogenous ALG3 gene is mutated, disrupted, truncated or partially deleted or completely deleted;
(Vi) comprises a polynucleotide encoding endomannosidase;
(Vii) one or more encoding a bifunctional UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase, N-acetylneuraminic acid-9-phosphate synthase or CMP-sialic acid synthase Contains a polynucleotide;
(Viii) the endogenous ATT1 gene is mutated, disrupted, truncated or partially deleted or completely deleted;
(Ix) the endogenous OCH1 gene is mutated, disrupted, truncated or partially deleted or completely deleted;
(X) comprising a polynucleotide encoding a galactosyltransferase;
(Xi) contains a nucleotide encoding a sugar nucleotide transporter;
(Xii) comprising a polynucleotide encoding a sialyltransferase;
(Xiii) include a polynucleotide encoding a acetylglucosaminyltransferase; and / or (xiv) one encoding flop protease or more endogenous genes mutation, disruption, truncated and partial deletion or total Has been deleted,
Isolated in any of claims 1 to 3 were Pichia pastoris (Pichia pastoris) host cell. 請 Motomeko isolated by Pichia pastoris one of 1 to 4 (Pichia pastoris) a host cell comprises a heterologous polynucleotide encoding a heterologous polypeptide, wherein the Pichia pastoris (Pichia pastoris) host cell. It said heterologous polypeptide is an immunoglobulin, isolated in any of claims 1 to 5 were Pichia pastoris (Pichia pastoris) host cell. (I) encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, and further comprising: L33 → STOP; Q214 → STOP; L294 → STOP; S298 → STOP; E403 → G; F406 → S; F406 → L: C411 → F; and selected from the group consisting of K469 → N, including one mutation , or
(Ii) comprising the nucleotide sequence of SEQ ID NO: 2 and further comprising a mutation selected from the group consisting of a1407c; g1232t: t1216c; t1217c; a1208g; c893a; t881g; c640t;
Isolated polynucleotide. An isolated vector comprising the polynucleotide of claim 7 . A method for producing an isolated by Pichia pastoris (Pichia pastoris) cells with a viability that was improved bioprocessing fermentation conditions:
(I) introducing a mutation into a gene encoding an endogenous CRZ1 polypeptide, wherein the mutation is selected from the group consisting of: producing a mutant CRZ1 polypeptide having one mutation; :
L294 → STOP; L294 → STOP; S298 → STOP; E403 → G; F406 → S; F406 → L: C411 → F; and K469 → N; or
(Ii) introducing mutations a1407c; g1232t: t1216c; t1217c; a1208g; c893a; t881g; c640t; or t98a into the polynucleotide.
Including
In said (i) or (ii), said mutation produces Pichia pastoris host cells with improved viability under biofermentation process conditions , wherein said improved viability is The method comprising a thermotolerant phenotype of surviving at 32 ° C. for about 90 to about 110 hours after methanol induction in a fermenter or bioreactor . An isolated fungal host cell produced by the method of claim 9 . The Pichia pastoris (Pichia pastoris) host cell, homologously recombined with the endogenous CRZ1 genes in the same cell, and the endogenous CRZ1 gene deleted partial deletion or total deletion, or destroy endogenous CRZ1 gene A method for producing an isolated Pichia pastoris host cell according to any of claims 1 to 6 , comprising introducing a heterologous polynucleotide. 12. An isolated Pichia pastoris host cell produced by the method of claim 11. (I) introducing a heterologous polynucleotide encoding said heterologous polypeptide into an isolated Pichia pastoris host cell of any of claims 1-4, 6, 10, and 12 ; and (ii) ) said Pichia pastoris (Pichia pastoris) culturing the Pichia pastoris (Pichia pastoris) host cell under conditions favoring expression of the heterologous polypeptide in the host cell and; further (iii) said Pichia pastoris (Pichia pastoris ) A method for producing one or more heterologous polypeptides, which may comprise isolating said heterologous polypeptide from a host cell. Conditions under which the heterologous polynucleotide encoding the heterologous polypeptide is operably linked to a methanol-inducible promoter and the isolated Pichia pastoris host cell is preferred for expression of the heterologous polypeptide in the presence of methanol 14. The method of claim 13 , wherein the method is cultured under.

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